Project Summary/Abstract The nasopharyngeal and respiratory mucosa represents a primary barrier to infection by inhaled organisms. However, many pathogens have adapted and can disseminate beyond the oral and respiratory mucosa to cause systemic disease. Such pathogens include Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumonia, and Bacillus anthracis. Together these pathogens account for millions of deaths worldwide every year, and in particular, M. tuberculosis continues to be a devastating global pathogen, alone causing two million deaths annually and latently infecting a third of the world?s population. Lining the respiratory mucosa are specialized epithelial cells that function to transcytose mucosal antigens from the apical, mucosal lumen to the basolateral space. These cells, known as microfold or M-cells, overlie mucosal associated lymphatic tissue where macrophages and dendritic cells await to ingest and present antigens to B-cells and T- cells. We hypothesized that M. tuberculosis has evolved to penetrate the mucosa via M-cells, and our preliminary data indicate that M. tuberculosis indeed utilizes M-cells to initiate disease. Blocking M-cell development or secretion of an M. tuberculosis virulence factor prevents lymph node dissemination and mortality from M. tuberculosis infection in mice. We will apply genetic, biochemical, immunologic and animal approaches to determine the functional role of M-cells in mucosal and systemic immunity against respiratory pathogens. Thus, in the proposed research we will (1) Characterize the transcytosis machinery leveraged by bacteria to cross airway M-cells, (2) Assess the role of airway M-cells in inducing innate and adaptive immunity in mice and (3) Determine the role of airway M-cells in the immune response using primary human tissues. The proposed work is expected to identify how M-cells bind and transcytose M. tuberculosis and similar bacteria, and the consequences of M-cell mediated transcytosis to innate and adaptive immune responses.